Luminescent metal complexes are valuable as probes and labels in a variety of applications such as diagnostic products and bioanalytical assay systems.
There is a continuing and expanding need for rapid, highly specific methods of detecting and quantifying chemical, biochemical and biological substances as analytes in research and diagnostic mixtures. Of particular value are methods for measuring small quantities of proteins, nucleic acids, peptides, pharmaceuticals, metabolites, microorganisms and other materials of diagnostic value. Examples of such materials include small molecular bioactive materials (e.g., narcotics and poisons, drugs administered for therapeutic purposes, hormones), pathogenic microorganisms and viruses, antibodies, and enzymes and nucleic acids, particularly those implicated in disease states.
The presence of a particular analyte can often be determined by binding methods that exploit the high degree of specificity, which characterizes many biochemical and biological systems. Frequently used methods are based on, for example, antigen-antibody systems, nucleic acid hybridization techniques, and protein-ligand systems. In these methods, the existence of a complex of diagnostic value is typically indicated by the presence or absence of an observable “label” which has been attached to one or more of the interacting materials. The specific labeling method chosen often dictates the usefulness and versatility of a particular system for detecting an analyte of interest. Preferred labels are inexpensive, safe, and capable of being attached efficiently to a wide variety of chemical, biochemical, and biological materials without significantly altering the important binding characteristics of those materials. The label should give a highly characteristic signal, and should be rarely, and preferably never, found in nature. The label should be stable and detectable in aqueous systems over periods of time ranging up to months. Detection of the label is preferably rapid, sensitive, and reproducible without the need for expensive, specialized facilities or the need for special precautions to protect personnel. Quantification of the label is preferably relatively independent of variables such as temperature and the composition of the mixture to be assayed.
A wide variety of labels have been developed, each with particular advantages and disadvantages. For example, radioactive labels are quite versatile, and can be detected at very low concentrations, such labels are, however, expensive, hazardous, and their use requires sophisticated equipment and trained personnel. Thus, there is wide interest in non-radioactive labels, particularly in labels that are observable by spectrophotometric, spin resonance, and luminescence techniques, and reactive materials, such as enzymes that produce such molecules.
Labels that are detectable using fluorescence spectroscopy are of particular interest, because of the large number of such labels that are known in the art. Moreover, the literature is replete with syntheses of fluorescent labels that are derivatized to allow their facile attachment to other molecules, and many such fluorescent labels are commercially available.
In addition to being directly detected, many fluorescent labels operate to quench or amplify the fluorescence of an adjacent second fluorescent label. Because of its dependence on the distance and the magnitude of the interaction between the quencher and the fluorophore, the quenching of a fluorescent species provides a sensitive probe of molecular conformation and binding, or other, interactions. An excellent example of the use of fluorescent reporter quencher pairs is found in the detection and analysis of nucleic acids.
An alternative detection scheme, which is theoretically more sensitive than autoradiography, is time-resolved fluorimetry. According to this method, a chelated lanthanide metal with a long radiative lifetime is attached to a molecule of interest. Pulsed excitation combined with a gated detection system allows for effective discrimination against short-lived background emission. For example, using this approach, the detection and quantification of DNA hybrids via an europium-labeled antibody has been demonstrated (Syvanen et al., Nucleic Acids Research 14: 1017-1028 (1986)). In addition, biotinylated DNA was measured in microtiter wells using Eu-labeled streptavidin (Dahlen, Anal. Biochem, 164: 78-83 (1982)). A disadvantage, however, of these types of assays is that the label must be washed from the probe and its fluorescence developed in an enhancement solution. A further drawback has been the fact that the fluorescence produced has only been in the nanosecond (ns) range, a generally unacceptably short period for adequate detection of the labeled molecules and for discrimination from background fluorescence.
In view of the predictable practical advantages it has been generally desired that the lanthanide chelates employed should exhibit a delayed luminescence with decay times of more than 10 μs. The fluorescence of many of the known fluorescent chelates tends to be inhibited by water and require augmentation with e.g. fluoride or micelles. As water is generally present in an assay, particularly an immunoassay system, lanthanide complexes that undergo inhibition of fluorescence in the presence of water are viewed as somewhat unfavorable or impractical for many applications. Moreover, the short fluorescence decay times is considered a disadvantage of these compounds. This inhibition is due to the affinity of the lanthanide ions for coordinating water molecules. When the lanthanide ion has coordinated water molecules, the absorbed light energy (excitation energy) is transferred from the complex to the solvent rather than being emitted as fluorescence.
Thus, stable lanthanide chelates, particularly coordinatively saturated chelates having excellent luminescence properties are highly desirable. In the alternative, coordinatively unsaturated lanthanide chelates that exhibit acceptable luminescence in the presence of water are also advantageous. Such chelates that are derivatized to allow their conjugation to one or more components of an assay, find use in a range of different assay formats. The present invention provides these and other such compounds and assays using these compounds.
Derivatives of 1-hydroxy-2-pyridinone (Structure 1) are of particular interest, since the ligand and its mono-anion (Structure 2) have a zwitterionic resonance form (Structure 3) that is isoelectronic with the catechol dianion.
Further, the 1-hydroxy-2-pyridinone structure possesses synthetic advantages, since the 6-carboxylic acid derivative (Structure 4) can be made in a straightforward manner.

Since the 1,2-HOPO ligands are useful sequestering agents for hard metal ions, especially for the f-elements, effort has been directed towards improving the initial synthesis of complexing ligands based on 1,2-HOPO. The original synthesis of multidentate 1,2-HOPO ligands, reported a decade ago, involves several low yield steps, difficult purifications and the use of phosgene (White et al., J. Med. Chem. 31: 11-18 (1988). The use of phosgene gas is undesirable for a number of reasons: the procedure is tedious; phosgene is highly toxic and volatile; the yield of the amine conjugate is low (e.g., yields of 3,4-LI-1,2-HOPO, and 3,4,3-LI-1,2-HOPO using phosgene were 34% and 15%, respectively); and the separation of the resulting product is difficult, often requiring the use of HPLC.
Uhlir reported that, following benzyl protection of the N-hydroxyl group of 6-carboxy-1,2-HOPO, this protected species could be activated and coupled to an amine scaffold (Uhlir, L. C. MIXED FUNCTIONALITY ACTINIDE SEQUESTERING AGENTS. Ph.D. thesis, University of California, Berkeley, 1992). Uhlir activated the HOPO carboxyl group using NHS/DCC and HOBT/DCC (see, Bodansky, M.; Bodanszky, A., THE PRACTICE OF PEPTIDE SYNTHESIS 2nd Ed., Springer-Verlag Berlin Heidelberg 1994, pp 96-125). Uhlir did not disclose the formation of an acid halide from the benzyl protected HOPO derivative.
Bailly et al. reported the multistep preparation of a benzyl protected 1,2-HOPO acid chloride and the use of the protected acid chloride to form amine conjugates of 1,2-HOPO (C. R. Acad. Sci. Paris 1, Serie II: 241-245 (1998)). The procedure of Bailly et al. is cumbersome, requiring conversion of the carboxylic acid to the corresponding methyl ester, activation and protection of the N-hydroxyl group, saponification of the methyl ester, followed by the activation of the carboxylic acid as the acid chloride. Bailly et al. does not suggest that the hydroxyl group can be protected in the presence of the free acid at the 6-position.
Other related art includes U.S. Pat. No. 4,698,431, which discloses polyvalent 1,2-HOPO chelators having an amide or a carboxylic acid moiety in the 6-position. The chelating agents are useful in selectively removing certain cations from solution and are particularly useful as ferric ion and actinide chelators. U.S. Pat. No. 5,892,029 and U.S. Pat. No. 5,624,901 also set forth polyvalent 1,2-HOPO chelators. None of the patents discloses or suggests preparing a polyvalent chelator from a protected, acid halide intermediate.
U.S. Pat. No. 4,666,927, discloses a number of chelating agents having 1,2-HOPO, 3,2-HOPO, or 3,4-HOPO moieties incorporated within their structures that are linked through a number of possible combinations of linking groups, including —CONH— groups. However, U.S. Pat. No. 4,666,927 teaches against a HOPO moiety having a substitution ortho to the hydroxy or oxo group of the HOPO ring, and does not disclose or suggest an acyl halide 1,2-HOPO intermediate.
A need for luminescent complexes, which are stable under biological relevant conditions and at low concentrations, remains. The current invention addresses these and other needs.